Nitric oxide (NO) is an important and well-characterized vasodilator essential in maintaining vascular tone in the systemic and cerebral circulation. The availability of NO is largely dependent upon three nitric oxide synthase (NOS) enzymes that catalyze L-arginine to citrulline, with the subsequent production of NO. We have recently shown that two specific variants in the promoter of the endothelial nitric oxide synthase (NOS3) gene (-922 G/A and -786 T/C) are associated with susceptibility to stroke in young, African-American women (Howard et al., Stroke, 2005). African-American women with the risk genotype for either of these two single nucleotide polymorphisms (SNPs) had approximately three times the risk of stroke compared to women with the non-risk genotypes (p = 0.005). This association was not observed in a similar group of Caucasian women, also taking part in the Stroke Prevention in Young Women (SPYW) study. We have recently performed a replication study in a group of women similarly ascertained and evaluated. With both promoter SNPs, evidence for replication of our original finding was observed in African-American women (p = 0.066 - 0.083). Borderline association was also observed in Caucasian women in this follow-up study (p = 0.058 - 0.071), although the association was in the opposite direction. While the NOS enzymes are directly responsible for the generation of NO, their activity is modulated by the availability of the substrate, L-arginine, and other regulators such as heat shock protein 90, calmodulin, and caveolin. The availability of L-arginine to cells, which is primarily obtained through dietary intake, is also determined by the arginine transporter (CAT1) and the activity of arginase, which catabolizes L-arginine to citrulline, with the production of urea, in the urea cycle. Therefore, the amount of NO produced at any given time is a direct result of the abundance and activity of the proteins and enzymes in this well-regulated pathway. Our previous results in the SPYW study and those of others investigating variability of NOS3 strongly implicate this pathway in the development of cerebro- and cardiovascular disease. Given the importance of NO in the regulation of vasomotor tone and the previously observed association of NOS3 SNPs with stroke in young African-American women, we hypothesize that polymorphisms in NOS3 and other genes affecting the generation and regulation of endothelial NO contribute to variability in endothelial function. This variability may lead to an increased risk of vascular diseases, such as ischemic stroke, in genetically susceptible individuals. The goal of this proposal is to evaluate variation in genes in the NO pathway and their relationship to ischemic stroke and subclinical endothelial dysfunction. Determining the genetic components of stroke susceptibility is an important step in delineating the pathogenesis of this disease. While environmental risk factors play an important role, genetic predisposition and gene-environment interactions represent important contributions that may increase our understanding of stroke and our ability to modify stroke risk and outcome. Collectively, the biological importance of NO and association studies with NOS3 strongly implicate NO generation and regulation as an important determinant of vascular disease. Enhanced understanding of the genetic regulation of the NO pathway may have important diagnostic and therapeutic value, leading to the identification of high-risk groups that might benefit from specific preventive interventions or to the development of new therapeutic drugs. [unreadable] [unreadable] [unreadable]